Getting More Resolution from Your LC Michael Woodman Columns and Consumables September 10, 2008

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1 Getting More Resolution from Your LC Michael Woodman Columns and Consumables September 10, 2008 Page 1

2 What are the Parameters Affecting Resolution? Isocratic Separations: physical and chemical separation parameters system dispersion (acts to degrade resolution) Gradient Separations physical and chemical separation parameters system dispersion gradient slope (effective k, aka k*) Page 2

3 Chromatographic Profile Equations Describing Factors Controlling R S Retention Factor k = (t r -t 0) t 0 Selectivity α = k2/k1 Theoretical Plates-Efficiency N = 5.54 (t r / w 1/2 ) 2 Page 3

4 The Logical Sequence to Improve Resolution k, α, N R s = k' k'+1 (α-1) α 4 N Retention Selectivity Theoretical Plates Page P2.PPT

5 Developing HPLC Separations Impact of k on the Resolution Equation R as Affected by Capacity R contribution (k'/(k'+1)) Capacity Factor (k') If k = 0, then R = 0 Page 5

6 Resolution as a Function of Selectivity, Column Efficiency, or Retention 7.00 Resolution Selectivity Impacts Resolution Most Change bonded phase chemistry Change mobile phase chemistry Change column temperature Increase N Increase Alpha Increase k' R s = N ½ /4 (α-1)/α k /(k +1) Plates: Alpha: k : Page 6

7 More RRHT Bonded Phase Choices Allow for Optimized Method Development mau Choices in SB family can be used for method optimization RRHT SB-Phenyl best choice with these conditions SB-C18 1.8um SB-C8 1.8um SB-Phenyl 1.8um SB-CN 1.8um Best Choice! Change in Elution Order Peaks 3,4 & 5 Sample: 1. Tolmetin 2. Naproxen 3. Diflunisal 4. Ibuprofen 5. Diclofenac For SB-Phenyl Conditions: Columns: RRHT 4.6 x 50mm, 1.8um Mobile Phase: Acetonitrile (40%):Buffer 60% ph=2.4 25mM NaH 2 PO 4 Flow Rate: 1.85 ml/ Detection: DAD, 254 nm LC: Agilent 1200 RR LC Sample: As listed, 1 ul injection Page 7

8 Broad Range of Agilent ZORBAX Selectivity -- Eclipse Plus Substituted for OEM Column Ethyl Hybrid C18, 4.6 x 150 mm, 5 um TF=1.25 TF= ZORBAX Eclipse Plus C18, 4.6 x 150 mm, 5 um TF=1.01 TF= Gradient: 10 to 30% B/15. A: 0.1% Formic Acid, B: 0.1% FA in ACN Flow: 1 ml/. Temperature 40 C Elution order: sulfanilamide, sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxazole Page 8

9 Temperature as an Aid to Method Development Temperature should always be considered as a parameter during method development Higher temperature provides more rapid mass transfer: Improves Efficiency enhances resolution, may reduce tailing Decreases analysis time lowers k Decreases Mobile Phase Viscosity Lowers backpressure allows for higher linear velocities, longer columns and smaller particle sizes Can change selectivity optimize resolution Page 9

10 Use Elevated Temperature to Optimize Resolution, Selectivity Gradient of Ten Cardiac Drugs on SB-C18 RRHT Rs= C Rs= C Rs= C Page 10

11 Increase Resolution by N (efficiency) When Do You Need It? How Do You Get It? When You have Complex Samples- Many Peaks, Limited Analysis Time Closely Related Compounds and. Changing the k has not helped Changing the Bonded Phase Has Not improved Resolution Changing the Mobile Phase Has Not Improved Resolution Temperature Has Not Helped What Remains That Will Improve Resolution? Page 11

12 Agilent ZORBAX RRHT Columns Provide Flexibility for More Resolution Column Length (mm) Resolving Power N(5 µm) Resolving Power N(3.5 µm) Resolving Power N(1.8 µm) Typical Pressure Bar (1.8 µm) Increase N by column L ,500 21,000 32, ,500 14,000 24, ,500 17, ,200 7,000 12, Increase N by particle size * Reduction in analysis time compared to 150 mm column pressure detered with 60:40 MeOH/water, 1ml/, 4.6mm ID Page 12

13 Agilent RRHT Columns -- No Selectivity Differences with Changes in Particle Sizes Under Same Conditions Column: 4.6 x 50mm Eclipse XDB-C18 Mobile Phase A= ph 4.5 Na Acetate, B=MeOH (60:40) Flow Rate =1 ml/ Inj. Vol: 2 ul Flow cell: 2uL, 3 mm flow path 5 µm t 0 =0.44 α 3,2 =1.20 α 4,3 =1.97 α 5,4 = Caffeine (I.S.) 2. Allobarbital 3. Phenobarbital 4. Butabarbital 5. Hexobarbital µm t 0 =0.44 α 3,2 =1.20 α 4,3 =1.97 α 5,4 = µm t 0 =0.46 α 3,2 =1.22 α 4,3 =1.94 α 5,4 = Page 13

14 RRHT 1.8um Columns Jan 1, 2008 Dimensions Eclipse Plus C18 Eclipse Plus C8 Eclipse XDB-C18 Eclipse XDB-C8 Extend-C18 Agilent Zorbax chemistries are available in a wide range of dimensions and particle sizes. 4.6 x x x x x x x x x x x x x x x Dimensions SB-C18 SB-C8 SB-Phenyl SB-CN SB-AQ Rx-Sil 4.6 x x x x x x x x x x x x x x x (Main product page) (P/N List) Page 14

15 Faster Analysis on Shorter Column with Smaller Particles, Resolution Conserved mau Original method ZORBAX LC column Extend-C x 150 mm, 5 μm 3 μl inj mau x faster ZORBAX RRHT column Extend C x 50 mm, 1.8 μm 1 μl inj Mobile phase: (70:30) MeOH: 50 mm pyrrolidine buffer Flow = 1.0 ml/, Temp. : ambient Page 15

16 1200 RRLC and RRHT Column Flexibility Conventional to High Speed HPLC Eclipse Plus C x 250 mm, 5 µm N(theory) 20,000 Conditions: A=pH 4.5 Na Acetate, B= Methanol (60:40) 1.0mL/ Flow cell: Micro/High Pressure 6mm, 1.7µL Detection: UV 254 nm Injection Volume: 2 ul Rapid Resolution Eclipse Plus C x 150 mm, 3.5 µm N(theory) 17, Rapid Resolution HT Eclipse Plus C x 50 mm, 1.8µm N(theory) 11, Sample: Phenobarbital 1. Caffeine 2. Allobarbital 3. Phenobarbital 4. Butabarbital 5. Hexobarbital Page 16

17 Page 17 ISOCRATIC ELUTION

18 Better Productivity High Resolution EPA 8330 Explosives Rapid Resolution Extend-C x 100 mm, 3.5 µm mau HMX RDX 135TNB 13DNB NB tetryl 2A DNT TNT 4A DNT Rs 7,6 : 1.3 Rs 8,7 :1.6 Rs 9,8 : DNT 26DNT 2NT 4NT 3NT PMP1, Pressure bar Mobile Phase: 75 A: 5mM NH4COOH (ph 6), 25 B: MeOH Column temp: 41C (temperature is critical in this separation) 2 ul injection x(0.5 ug ea/ml) Flow: 2.5 ml/. Extend-C18 provides greater resolution of explosives in EPA 8330 than the two column method. This dramatically improves the Greening of the lab! Page 18

19 Sharpen Late Eluting Peaks Lower Flow with Gradient Bump 4.6 x 100 mm, 3.5 µm Extend-C18 mau DAD1 B, Sig=250,60 Ref=360,100 (070803\EXTEND_3X D) HMX 135TNB RDX 13DNB NB tetryl 4A DNT TNT 4NT 2A DNT 24 DNT 26DNT 2NT 3NT bar PMP1, Pressure MP:75 A: 5mM ammonium formate, ph 4.9 w formic acid, 25 B: MeOH At 14 B=40% Flow: 1.7 ml/. temp: 40C 2 ul injection x(50ug ea/ml) Max press is only 210 bar Page 19

20 Sharper Peaks - Higher Resolution 4.6 x 100 mm, 1.8µm Extend-C18 mau 40 DAD1 B, Sig=250,60 Ref=360,100 (070803\EXTEND_3X D) bar PMP1, Pressure MP: 75 A: 5mM NH4COOH (ph 6), 25 B: MeOH At 15 B=40% Flow: 1.7 ml/. temp: 40C 2 ul injection x(50ug ea/ml) Page 20

21 Page 21 GRADIENT ELUTION

22 Column Efficiency under Gradient Conditions Concept of Peak Capacity. Standard concept of column efficiency, N (plates), is only appropriate for isocratic conditions. A more useful concept for the case of gradient conditions is Peak Capacity - the number of peaks that can be separated (at a specified resolution) in a given amount of time. It is another measure of column efficiency. 4 P c = (1+ t G /w) for gradient conditions 5 μm 1.8 μm 5 2 peaks fit Rs = +50% 3 peaks fit 50% more!! Page 22

23 Resolution Improvements on Long 1.8 μm Columns 2.1 x150mm SB-C18 RRHT - 5µm vs. 1.8µm mau um vs. 5um + 60% Resolution (Theory: 67%) + 160% Efficiency (Theory: 177%) Pressure at start: 51bar, 5µm Rs(4,5) = 3.14 Peak Width 5σ (9) = Pressure at start: 394bar, 1.8µm Rs(4,5) = 5.07 Peak Width 5σ (9) = Resolution gains are independent of column ID on a low volume LC Page 23

24 Ginseng Extract mau mau Agilent 1100 Series 400 bar Agilent 1200 Series 600 bar Column: 2.1x150 mm Zorbax SB C-18, 5 µm µm Column: 2.1x150 mm Zorbax SB C-18, 1.8 µm 1.8 µm Mobile phases: Gradient: Flow: Inj. volume: DAD: A = H2O + 0.1% TFA B = ACN + 0.1% TFA 10% to 95% ACN in 40, hold for ml/ 3 μl, partial loop filling 220 nm (20 Hz) 2µl flow cell Improved Resolution Agilent Application Note: Analysis of a complex natural product extract from ginseng Part I Publication Number EN Page 24

25 100% B t g = 5 Relationship of k* and Key Gradient Parameters 0% B 100% B k* t g F 0% B t g = % B S Δ%B V m 1/k* = gradient steepness = b 0% B t g = 20 t g = % B ΔΦ = solvent S = F = t = g V m = change in volume fraction of B constant (molecular weight dependent) flow rate (ml/.) gradient time (.) column void volume (ml) 0% B Time () P1.PPT Group/Presentation Title Agilent Restricted Month ##, 200X

26 Improving Resolution Using Gradient Slope N (theory) 12,000 N (theory) 5,700 k* (estimated) 4.5 k* (estimated) SB-C8, 4.6 x 150 mm, 5µm 300SB-C8, 4.6 x 50 mm, 3.5µm Mobile Phase: Flow Rate: Temperature: A: 95% Water : 5 % ACN, 0.1% TFA B: 5% Water : 95% ACN, 0.085% TFA Gradient: 10-60% B in 30. Sample: 1. Gly-Tyr 2. Val-Tyr-Val 3. [Gln 11 ] Amyloid-β- 6. Leu-Enk 7. Angiotensin II 8. Kinetensin 1.0 ml /. Protein Fragm RNase Ambient 4. (TYR8) Bradykinin 10. Insulin (Eq.) 5. Met-Enk Page 26

27 Peptide Map of Tryptic Digest of BSA More Peak Capacity an Rs Zorbax SB-C18, 2.1x100mm, 1.8µ mau MWD1 A, Sig=212,4 Ref=350,20 (NEWDIGEST X150U\PEPM APNEWT RYP27.D) Gradient time Peak Capacity mau MWD1 A, Sig=212,4 Ref=350,20 (NEWDIGEST X150U\PEPM APNEWT RYP31.D) mau MWD1 A, Sig=212,4 Ref=350,20 (NEWDIGEST X150U\PEPM APNEWT RYP35.D) % increase in peak capacity as t g extended Page 27

28 Optimizing the 1200 Rapid Resolution System: Your Guide to Achieving High Resolution Separations Page 28

29 High Resolution Chromatography Important aspects & frequently asked questions Delay/dwell volume How do I utilize the configurable delay volume? Extra column volume How should I stack my 1200 RRLC? Green, Red, or Natural PEEK- what tubing should I use? Detector Parameters Data Acquisition Rate Flow Cell Volume Calculate before you run... Extracolumn volume Delay/Dwell volume Page 29

30 Why is delay/dwell volume important? 1. Different dwell volumes result in a RT time shift (the time for the mobile phase to reach the column head) 2. Different dwell volume could effect resolution (peaks spends different time under isocratic/gradient conditions) 3. The dwell volume is important for narrow bore applications with low flow rates Example: mm ID column, flow rate: 0.5 ml/ A delay volume of 1 ml will result in a RT shift of 2-3 mm ID column, flow rate: 4 ml/ A delay volume of 1 ml will result in RT shift of 15 sec Page 30

31 Various Delays, Binary 1200 Liftoff and midpoint delays vary widely 50% 1100 Binary/WPS, as shipped with mixer 1100 Binary/WPS, mixer removed 1100 Binary/WPS, mixer removed, w/ ADVR 1100 Binary/WPS, mixer removed, ADVR, PD moved Flow=500ul/ Step at inject 70 bar restrictor The pd is moved to the A channel, between the outlet check valve and the mixing T Page 31

32 Agilent HPLC Delay Volumes 1200 (not SL) Binary WPS Configurations Configuration Approximate Liftoff volume Approximate Midpoint vol. As shipped 675ul 975ul Remove mixer 375ul 525ul relocated pulse damper 275ul 390ul and use ADVR 100ul 180ul Page 32

33 Delay/dwell volumes, 1200 RRLC System (1200SL) How do I utilize the configurable delay volume? Std delay volume µl typical 3 and 4.6 mm ID columns and conventional LC Pressure Sensor A 400µl Mixer Purge Valve B 600bar Damper Mixing-T Pressure-Sensor Damper 400µl Mixer Purge Valve Low delay volume 120 µl ballistic LC with low volume (usually 2.1 mm ID) columns Pressure Sensor A 400µl Mixer Purge Valve 600bar Damper B Flow Path Mixing-T Pressure-Sensor Purge Valve Page 33

34 More 1200SL options: 200µl Mixer Medium delay volume 320 µl as needed to improve mixing efficiency (TFA in the mobile phase with a low UV wavelength, for example) Pressure Sensor A Purge Valve 600bar Damper B Mixing-T Pressure-Sensor 200µl Mixer Purge Valve Switching between configuration: manually by dis-/re- connecting of capillaries automatically by using a 2PS/6PT valve Page 34

35 Mainpass/Bypass (ADVR) Reduces Delay Main Pass Bypass Page 35

36 Loss of Efficiency due to Dispersion Peak Volumes vs. Column Dimensions, Particle Size and Capacity Factors (k ) Guidelines for selecting flow cells, and raising awareness of various factors contributing to bandspreading and loss of resolution in HPLC separations Page 36

37 Extra-Column Volume What is the problem? 10 µl Sample Volume Volume of Connective Tubing Detector Flow Cell Volume Detector Electronic Time Constant 20 µl System dispersion effects often over-shadow changes in particle size, especially with smaller columns Time,. Page 37

38 Extra column volumes How should I stack my 1200 RRLC? Single Stack: Optimize for imal lab bench foot-print, shortest capillary lengths: Gradient Delay Volume Extracolumn Dispersion Shown: Single stack configuration equipped with low dispersion volume capillaries ( 0.12 mm id - red) Page 38

39 Flow Cell Properties, Impact on Small Columns mm Cell Pathlength ul Cell Volume Relative Sensitivity Measured Dispersion - System Loss of N* (2.1x50, 1.8um, k =10) % % % 10 nano.5?? (higher noise) ~9 27% *Loss of N vs. theory with 0 dispersion. System contribution included. Page 39

40 Influence of the detector rate on resolution DAD SL and MWD SL PW=0.30sec PW=0.33sec 80Hz 80Hz versus 10Hz Data Rate Peak Width: 55% Resolution: + 90% Peak Capacity: + 120% App. Column Eff.: + 260% PW=0.42sec 40Hz Data Rate Peak Width Resolution Peak Capacity 80 Hz Hz PW=0.67sec PW=1.24sec 20Hz 10Hz 5Hz Hz Hz Hz Sample: Column: Gradient:: Flow Rate: Phenones Test Mix Zorbax SB-C18, 4.6x30, 1.8um %ACN in 0.3 5ml/ Page 40

41 k vs. column dimensions vs. peak volume Peak Volume vs Col Vol k' 10 (blue), k'2 (pink) 0.5 Peak Volume (Disp=10ul) mm k 10 3mm columns k Column Bed Volume 4.6mm columns, k 10 (30,50,100, 150mm) 4.6, 3 and 2.1mm columns, k 2 Series 1 Series 2 Page 41

42 Calculated numbers for N 100mm 1.8um columns, k*= N estimated, 5 sigma mm 3.0mm 2.1mm 1.0mm Series1 Series2 Series3 Series4 dispersion, 5 sigma Page 42

43 Yield estimates 1.8um, 2.1, 3.0, 4.6mm i.d. with low and high dispersion Yield % of N vs Dispersion Calculated Yield of N 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 3.0 x 50, 100, 150mm 2.1 x 50, 100, 150mm 4.6 x 50, 100, 150mm ul dispersion, 1.8um particle size Calculated Column Bed Volume 8ul dispersion, 1.8um particle size Page 43

44 Conclusion What about dispersion? There is no simple method to suggest the flow cell selection Increased dispersion due to flow cell volume must be weighed against the benefit of longer pathlength and increased sensitivity The method converter is an appropriate tool for making these estimates There is no question that overall dispersion must be imized, appropriately, regardless of the column dimensions. Page 44

45 The Agilent 1200 LC Systems One Comprehensive Series of Solutions for your Applications Application 1200 RR TM LC 1100/1200 Binary 1100/1200 Quaternary RRLC Narrow (2.1) Ultra-fast column length 15 50mm High-resolution column length mm YES YES NO Delay volume too large NO Delay volume too large, pressure limit too low NO Delay volume too large NO Delay volume too large, pressure limit too low RRLC Std (3.0, 4.6) Ultra-fast column length 15 50mm YES YES YES Modifications recommended to reduce delay volume High-resolution column length mm YES NO Pressure limit too low NO Pressure limit too low HPLC Narrow bore (2.1) YES YES Modifications recommended to reduce delay volume NO Delay volume too large HPLC Standard bore (3.0, 4.6) YES YES YES Page 45

46 Explore New Conditions at Your Desk Simple and fast with the new Agilent Method Translator 1. Input Values 2. Conversion: Conventional HPLC method CONVERT Optimized RRLC method Page 46

47 RRLC-Method: Final optimized Method Agilent Cost Savings Calculator Enter General Settings & Instrument Settings Calculated Annual Savings (Laboratory View) (Financial View) Page 47

48 Literature Technical Notes & Applications Easy Transfer of Standard HPLC Methods to the Agilent 1200 Series Rapid Resolution LC System Publication Number EN Seamless Transfer of Methods to the New Agilent 1200 Series Diode Array Detector Publication Number EN Upgrading an Agilent 1100 Series LC system to an Agilent 1200 Series Rapid Resolution LC system for higher performance Part 1: Optimizing for use with 2.1 mm ID columns Part 2: Optimizing for use with 4.6 mm ID columns Publication Numbers EN, EN Agilent 1200 Series Rapid Resolution System Compendium CD Publication Number EN Latest information on pharmaceutical applications and monthly compliance article by Dr. Ludwig Huber: Page 48

49 Summary Resolution can be improved with careful optimization of separation selectivity and efficiency Systems can be optimized to deliver appropriate dwell volume for the application requirements System dispersion degrades all separations and must be measured and reduced whenever practical Use method optimization tools to make good decisions before making significant system changes and/or purchasing new columns. Saves time and money. Page 49

50 R1669A RRLC: Method Development and Optimization Course Features: Two day hands-on course with lab sessions Rapid resolution fundamentals overview Hardware overview Column selection Influence of key chromatographic parameters to RRLC Who Would Benefit from this Course: This course will help users who are responsible for method development or method optimization needing to reduce sample run times or increase sample throughput by applying rapid resolution technology in their lab. Further details are available at: Click on Education & Events to view the calendar and register on-line or call Page 50

51 Upcog LC esears Getting the Most from your LC Optimizing for Speed September 11, :00pm EDT Improving HPLC Selectivity and Resolution for Protein and Peptide Separations September 30, :00pm EDT Discovering and Identifying More Protein Targets in Less Time October 21, :00pm EDT Page 51

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